Miroslav Valarik - Academia.edu (original) (raw)
Papers by Miroslav Valarik
Proceedings of The National Academy of Sciences, 2006
Winter wheat and barley varieties require an extended exposure to low temperatures to accelerate ... more Winter wheat and barley varieties require an extended exposure to low temperatures to accelerate flowering (vernalization), whereas spring varieties do not have this requirement. In this study, we show that in these species, the vernalization gene VRN3 is linked completely to a gene similar to Arabidopsis FLOWERING LOCUS T (FT). FT induction in the leaves results in a transmissible signal that promotes flowering. Transcript levels of the barley and wheat orthologues, designated as HvFT and TaFT, respectively, are significantly higher in plants homozygous for the dominant Vrn3 alleles (early flowering) than in plants homozygous for the recessive vrn3 alleles (late flowering). In wheat, the dominant Vrn3 allele is associated with the insertion of a retroelement in the TaFT promoter, whereas in barley, mutations in the HvFT first intron differentiate plants with dominant and recessive VRN3 alleles. Winter wheat plants transformed with the TaFT allele carrying the promoter retroelement insertion flowered significantly earlier than nontransgenic plants, supporting the identity between TaFT and VRN-B3. Statistical analyses of flowering times confirmed the presence of significant interactions between vernalization and FT allelic classes in both wheat and barley (P < 0.0001). These interactions were supported further by the observed up-regulation of HvFT transcript levels by vernalization in barley winter plants (P ؍ 0.002). These results confirmed that the wheat and barley FT genes are responsible for natural allelic variation in vernalization requirement, providing additional sources of adaptive diversity to these economically important crops. flowering ͉ Triticum aestivum ͉ Flowering Locus T ͉ Hordeum vulgare Abbreviations: LD, long day; QTL, quantitative trait loci; RFLP, restriction fragment length polymorphism; RSL, recombinant substitution line; SD, short day. † Present address:
Molecular and General Genetics, 2000
Retroelements are ubiquitous features of eukaryotic genomes, often accounting for a substantial f... more Retroelements are ubiquitous features of eukaryotic genomes, often accounting for a substantial fraction of their total DNA content. One major group of retroelements, which includes the gypsy and copia-like elements, is distinguished by the presence of long terminal repeats (LTRs). We have identified and partially characterized a sequence from banana (Musa acuminata cv. Grand Nain) which shows significant homology to gypsy-like LTR retroelements from other species. The element, named monkey, shows a high degree of homology to the reverse transcriptase, RNase H and integrase genes of retroelements from plants, fungi and yeast. However, several stop codons are present in the major ORF of this element, suggesting that this copy of monkey, if functional, is non-autonomous. Southern analysis indicated that monkey is present in both the A and B genomes of Musa, and that it is found in 200–500 copies per haploid genome in cv. Grand Nain. Chromosomal localization by fluorescent in-situ hybridization indicates that copies of monkey are concentrated in the nucleolar organizer regions and colocalize with rRNA genes. Other copies of monkey appear to be dispersed throughout the genome.
Theoretical and Applied Genetics, 2004
The analysis of the hexaploid wheat genome (Triticum aestivum L., 2n=6x=42) is hampered by its la... more The analysis of the hexaploid wheat genome (Triticum aestivum L., 2n=6x=42) is hampered by its large size (16,974 Mb/1C) and presence of three homoeologous genomes (A, B and D). One of the possible strategies is a targeted approach based on subgenomic libraries of large DNA inserts. In this work, we purified by flow cytometry a total of 107 of three wheat D-genome chromosomes: 1D, 4D and 6D. Chromosomal DNA was partially digested with HindIII and used to prepare a specific bacterial artificial chromosome (BAC) library. The library (designated as TA-subD) consists of 87,168 clones, with an average insert size of 85 kb. Among these clones, 53% had inserts larger than 100 kb, only 29% of inserts being shorter than 75 kb. The coverage was estimated to be 3.4-fold, giving a 96.5% probability of identifying a clone corresponding to any sequence on the three chromosomes. Specificity for chromosomes 1D, 4D and 6D was confirmed after screening the library pools with single-locus microsatellite markers. The screening indicated that the library was not biased and gave an estimated coverage of sixfold. This is the second report on BAC library construction from flow-sorted plant chromosomes, which confirms that dissecting of the complex wheat genome and preparation of subgenomic BAC libraries is possible. Their availability should facilitate the analysis of wheat genome structure and evolution, development of cytogenetic maps, construction of local physical maps and map-based cloning of agronomically important genes.
Plant Journal, 2004
The analysis of the complex genome of common wheat (Triticum aestivum, 2n ¼ 6x ¼ 42, genome formu... more The analysis of the complex genome of common wheat (Triticum aestivum, 2n ¼ 6x ¼ 42, genome formula AABBDD) is hampered by its large size ($17 000 Mbp) and allohexaploid nature. In order to simplify its analysis, we developed a generic strategy for dissecting such large and complex genomes into individual chromosomes. Chromosome 3B was successfully sorted by flow cytometry and cloned into a bacterial artificial chromosome (BAC), using only 1.8 million chromosomes and an adapted protocol developed for this purpose. The BAC library (designated as TA-3B) consists of 67 968 clones with an average insert size of 103 kb. It represents 6.2 equivalents of chromosome 3B with 100% coverage and 90% specificity as confirmed by genetic markers. This method was validated using other chromosomes and its broad application and usefulness in facilitating wheat genome analysis were demonstrated by target characterization of the chromosome 3B structure through cytogenetic mapping. This report on the successful cloning of flow-sorted chromosomes into BACs marks the integration of flow cytogenetics and genomics and represents a great leap forward in genetics and genomic analysis.
Chromosome Research, 2002
Partial genomic DNA libraries were constructed in Musa acuminata and M. balbisiana and screened f... more Partial genomic DNA libraries were constructed in Musa acuminata and M. balbisiana and screened for clones carrying repeated sequences, and sequences carrying rDNA. Isolated clones were characterized in terms of copy number, genomic distribution in M. acuminata and M. balbisiana, and sequence similarity to known DNA sequences. Ribosomal RNA genes have been the most abundant sequences recovered. FISH with probes for DNA clones Radka1 and Radka7, which carry different fragments of Musa 26S rDNA, and Radka14, for which no homology with known DNA sequences has been found, resulted in clear signals at secondary constrictions. Only one clone carrying 5S rDNA, named Radka2, has been recovered. All remaining DNA clones exhibited more or less pronounced clustering at centromeric regions. The study revealed small differences in genomic distribution of repetitive DNA sequences between M. acuminata and M. balbisiana, the only exception being the 5S rDNA where the two Musa clones under study differed in the number of sites. All repetitive sequences were more abundant in M. acuminata whose genome is about 12% larger than that of M. balbisiana. While, for some sequences, the differences in copy number between the species were relatively small, for some of them, e.g. Radka5, the difference was almost thirty-fold. These observations suggest that repetitive DNA sequences contribute to the difference in genome size between both species, albeit to different extents. Isolation and characterization of new repetitive DNA sequences improves the knowledge of long-range organization of chromosomes in Musa.
Biologia Plantarum, 1998
Fluorescent in situ hybridisation (FISH) was used to determine the number and distribution of the... more Fluorescent in situ hybridisation (FISH) was used to determine the number and distribution of the 18S-25S and 5S rDNA sites on mitotic chromosomes of 6 wild and 2 edible diploid (2n=22) accessions belonging to the two banana species, Musa acuminata and M. balbisiana. FISH with the 18S-25S probe resulted in signals on one pair of chromosomes, the position of signals corresponded to the secondary constriction at the end of a short arm. The intensity of labelling was different between the homologues and the larger site corresponded to a larger secondary constriction. This labelling pattern was observed consistently in all genotypes. On the other hand, differences in the number of 5S sites were observed between the accessions. While in some of the wild seeded species, the 5S rDNA was localised on two pairs of chromosomes, hybridisation signals appeared on three pairs of chromosomes in other wild accessions. Quite unexpectedly, only five sites of 5S rDNA were reproducibly observed in the two vegetatively propagated diploid edible cultivars, Pisang Mas and Niyarma Yik, evidence for structural heterozygosity. A dual colour FISH showed that in all accessions, the satellite chromosomes carrying the 18S-25S loci did not carry the 5S loci. The results demonstrate that molecular cytogenetics can be applied to Musa and that physical cytogenetic maps can be generated.
The Triticeae species are unique among the important agricultural crops in possessing massive gen... more The Triticeae species are unique among the important agricultural crops in possessing massive genomes with a prevalence of dispersed DNA repeats. The highest level of complexity is observed in tetraploid and hexaploid wheat whose nuclear genomes comprise two and three homoeologous genomes, respectively. Polyploidy and the presence of repeats make gene cloning and genome sequencing in the Triticeae extremely difficult. Chromosome genomics simplifies these tasks by targeting single chromosomes and chromosome arms, which represent only a few percent of the nuclear genomes. The advantages of this strategy over a whole-genome approach include the avoidance of problems due to the presence of homoeologs in wheat, reduction of work to manageable portions, cost efficiency, and an opportunity to structure collaborative projects where individual laboratories work on particular chromosomes. In this chapter, we describe how chromosomes and chromosome arms can be isolated by flow cytometric sorting and we review development of flow cytogenetics in the Triticeae. We then discuss various applications of flow-sorted chromosomes and assess the potential of chromosome genomics in the Triticeae.
BMC Evolutionary Biology, 2011
Background The classification of the Musaceae (banana) family species and their phylogenetic inte... more Background The classification of the Musaceae (banana) family species and their phylogenetic inter-relationships remain controversial, in part due to limited nucleotide information to complement the morphological and physiological characters. In this work the evolutionary relationships within the Musaceae family were studied using 13 species and DNA sequences obtained from a set of 19 unlinked nuclear genes. Results The 19 gene sequences represented a sample of ~16 kb of genome sequence (~73% intronic). The sequence data were also used to obtain estimates for the divergence times of the Musaceae genera and Musa sections. Nucleotide variation within the sample confirmed the close relationship of Australimusa and Callimusa sections and showed that Eumusa and Rhodochlamys sections are not reciprocally monophyletic, which supports the previous claims for the merger between the two latter sections. Divergence time analysis supported the previous dating of the Musaceae crown age to the Cretaceous/Tertiary boundary (~ 69 Mya), and the evolution of Musa to ~50 Mya. The first estimates for the divergence times of the four Musa sections were also obtained. Conclusions The gene sequence-based phylogeny presented here provides a substantial insight into the course of speciation within the Musaceae. An understanding of the main phylogenetic relationships between banana species will help to fine-tune the taxonomy of Musaceae.
Genome, 2003
Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were deve... more Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were developed for rye (Secale cereale L.). Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity obtained after the analysis of DAPI-stained chromosomes (flow karyotypes) were characterized and the chromosome content of the DNA peaks was determined. Chromosome 1R could be discriminated on a flow karyotype of S. cereale 'Imperial'. The remaining rye chromosomes (2R-7R) could be discriminated and sorted from individual wheat-rye addition lines. The analysis of lines with reconstructed karyotypes demonstrated a possibility of sorting translocation chromosomes. Supernumerary B chromosomes could be sorted from an experimental rye population and from S. cereale 'Adams'. Flow-sorted chromosomes were identified by fluorescence in situ hybridization (FISH) with probes for various DNA repeats. Large numbers of chromosomes of a single type sorted onto microscopic slides facilitated detection of rarely occurring chromosome variants by FISH with specific probes. PCR with chromosome-specific primers confirmed the identity of sorted fractions and indicated suitability of sorted chromosomes for physical mapping. The possibility to sort large numbers of chromosomes opens a way for the construction of large-insert chromosome-specific DNA libraries in rye.
Plant Journal, 2004
A novel high-resolution fluorescence in situ hybridisation (FISH) strategy, using super-stretched... more A novel high-resolution fluorescence in situ hybridisation (FISH) strategy, using super-stretched flow-sorted plant chromosomes as targets, is described. The technique that allows longitudinal extension of chromosomes of more than 100 times their original metaphase size is especially attractive for plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves flow cytometric sorting of metaphase chromosomes, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol:acetic acid (3 : 1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea, hybridised with 45S and 5S ribosomal DNAs and the [GAA]n microsatellite, the [TTTAGGG]n telomeric repeat and a bacterial artificial chromosome (BAC) clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity of 1 kbp. Spatial resolution of neighbouring loci was improved down to 70 kbp as compared to 5–10 Mbp after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA fibres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is confined to species for which chromosome flow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.
A novel high-resolution¯uorescence in situ hybridisation (FISH) strategy, using super-stretched¯o... more A novel high-resolution¯uorescence in situ hybridisation (FISH) strategy, using super-stretched¯owsorted plant chromosomes as targets, is described. The technique that allows longitudinal extension of chromosomes of more than 100 times their original metaphase size is especially attractive for plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves¯ow cytometric sorting of metaphase chromosomes, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol:acetic acid (3 : 1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea, hybridised with 45S and 5S ribosomal DNAs and the [GAA] n microsatellite, the [TTTAGGG] n telomeric repeat and a bacterial arti®cial chromosome (BAC) clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity of 1 kbp. Spatial resolution of neighbouring loci was improved down to 70 kbp as compared to 5±10 Mbp after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA ®bres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is con®ned to species for which chromosome¯ow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.
Proceedings of The National Academy of Sciences, 2006
Winter wheat and barley varieties require an extended exposure to low temperatures to accelerate ... more Winter wheat and barley varieties require an extended exposure to low temperatures to accelerate flowering (vernalization), whereas spring varieties do not have this requirement. In this study, we show that in these species, the vernalization gene VRN3 is linked completely to a gene similar to Arabidopsis FLOWERING LOCUS T (FT). FT induction in the leaves results in a transmissible signal that promotes flowering. Transcript levels of the barley and wheat orthologues, designated as HvFT and TaFT, respectively, are significantly higher in plants homozygous for the dominant Vrn3 alleles (early flowering) than in plants homozygous for the recessive vrn3 alleles (late flowering). In wheat, the dominant Vrn3 allele is associated with the insertion of a retroelement in the TaFT promoter, whereas in barley, mutations in the HvFT first intron differentiate plants with dominant and recessive VRN3 alleles. Winter wheat plants transformed with the TaFT allele carrying the promoter retroelement insertion flowered significantly earlier than nontransgenic plants, supporting the identity between TaFT and VRN-B3. Statistical analyses of flowering times confirmed the presence of significant interactions between vernalization and FT allelic classes in both wheat and barley (P < 0.0001). These interactions were supported further by the observed up-regulation of HvFT transcript levels by vernalization in barley winter plants (P ؍ 0.002). These results confirmed that the wheat and barley FT genes are responsible for natural allelic variation in vernalization requirement, providing additional sources of adaptive diversity to these economically important crops. flowering ͉ Triticum aestivum ͉ Flowering Locus T ͉ Hordeum vulgare Abbreviations: LD, long day; QTL, quantitative trait loci; RFLP, restriction fragment length polymorphism; RSL, recombinant substitution line; SD, short day. † Present address:
Molecular and General Genetics, 2000
Retroelements are ubiquitous features of eukaryotic genomes, often accounting for a substantial f... more Retroelements are ubiquitous features of eukaryotic genomes, often accounting for a substantial fraction of their total DNA content. One major group of retroelements, which includes the gypsy and copia-like elements, is distinguished by the presence of long terminal repeats (LTRs). We have identified and partially characterized a sequence from banana (Musa acuminata cv. Grand Nain) which shows significant homology to gypsy-like LTR retroelements from other species. The element, named monkey, shows a high degree of homology to the reverse transcriptase, RNase H and integrase genes of retroelements from plants, fungi and yeast. However, several stop codons are present in the major ORF of this element, suggesting that this copy of monkey, if functional, is non-autonomous. Southern analysis indicated that monkey is present in both the A and B genomes of Musa, and that it is found in 200–500 copies per haploid genome in cv. Grand Nain. Chromosomal localization by fluorescent in-situ hybridization indicates that copies of monkey are concentrated in the nucleolar organizer regions and colocalize with rRNA genes. Other copies of monkey appear to be dispersed throughout the genome.
Theoretical and Applied Genetics, 2004
The analysis of the hexaploid wheat genome (Triticum aestivum L., 2n=6x=42) is hampered by its la... more The analysis of the hexaploid wheat genome (Triticum aestivum L., 2n=6x=42) is hampered by its large size (16,974 Mb/1C) and presence of three homoeologous genomes (A, B and D). One of the possible strategies is a targeted approach based on subgenomic libraries of large DNA inserts. In this work, we purified by flow cytometry a total of 107 of three wheat D-genome chromosomes: 1D, 4D and 6D. Chromosomal DNA was partially digested with HindIII and used to prepare a specific bacterial artificial chromosome (BAC) library. The library (designated as TA-subD) consists of 87,168 clones, with an average insert size of 85 kb. Among these clones, 53% had inserts larger than 100 kb, only 29% of inserts being shorter than 75 kb. The coverage was estimated to be 3.4-fold, giving a 96.5% probability of identifying a clone corresponding to any sequence on the three chromosomes. Specificity for chromosomes 1D, 4D and 6D was confirmed after screening the library pools with single-locus microsatellite markers. The screening indicated that the library was not biased and gave an estimated coverage of sixfold. This is the second report on BAC library construction from flow-sorted plant chromosomes, which confirms that dissecting of the complex wheat genome and preparation of subgenomic BAC libraries is possible. Their availability should facilitate the analysis of wheat genome structure and evolution, development of cytogenetic maps, construction of local physical maps and map-based cloning of agronomically important genes.
Plant Journal, 2004
The analysis of the complex genome of common wheat (Triticum aestivum, 2n ¼ 6x ¼ 42, genome formu... more The analysis of the complex genome of common wheat (Triticum aestivum, 2n ¼ 6x ¼ 42, genome formula AABBDD) is hampered by its large size ($17 000 Mbp) and allohexaploid nature. In order to simplify its analysis, we developed a generic strategy for dissecting such large and complex genomes into individual chromosomes. Chromosome 3B was successfully sorted by flow cytometry and cloned into a bacterial artificial chromosome (BAC), using only 1.8 million chromosomes and an adapted protocol developed for this purpose. The BAC library (designated as TA-3B) consists of 67 968 clones with an average insert size of 103 kb. It represents 6.2 equivalents of chromosome 3B with 100% coverage and 90% specificity as confirmed by genetic markers. This method was validated using other chromosomes and its broad application and usefulness in facilitating wheat genome analysis were demonstrated by target characterization of the chromosome 3B structure through cytogenetic mapping. This report on the successful cloning of flow-sorted chromosomes into BACs marks the integration of flow cytogenetics and genomics and represents a great leap forward in genetics and genomic analysis.
Chromosome Research, 2002
Partial genomic DNA libraries were constructed in Musa acuminata and M. balbisiana and screened f... more Partial genomic DNA libraries were constructed in Musa acuminata and M. balbisiana and screened for clones carrying repeated sequences, and sequences carrying rDNA. Isolated clones were characterized in terms of copy number, genomic distribution in M. acuminata and M. balbisiana, and sequence similarity to known DNA sequences. Ribosomal RNA genes have been the most abundant sequences recovered. FISH with probes for DNA clones Radka1 and Radka7, which carry different fragments of Musa 26S rDNA, and Radka14, for which no homology with known DNA sequences has been found, resulted in clear signals at secondary constrictions. Only one clone carrying 5S rDNA, named Radka2, has been recovered. All remaining DNA clones exhibited more or less pronounced clustering at centromeric regions. The study revealed small differences in genomic distribution of repetitive DNA sequences between M. acuminata and M. balbisiana, the only exception being the 5S rDNA where the two Musa clones under study differed in the number of sites. All repetitive sequences were more abundant in M. acuminata whose genome is about 12% larger than that of M. balbisiana. While, for some sequences, the differences in copy number between the species were relatively small, for some of them, e.g. Radka5, the difference was almost thirty-fold. These observations suggest that repetitive DNA sequences contribute to the difference in genome size between both species, albeit to different extents. Isolation and characterization of new repetitive DNA sequences improves the knowledge of long-range organization of chromosomes in Musa.
Biologia Plantarum, 1998
Fluorescent in situ hybridisation (FISH) was used to determine the number and distribution of the... more Fluorescent in situ hybridisation (FISH) was used to determine the number and distribution of the 18S-25S and 5S rDNA sites on mitotic chromosomes of 6 wild and 2 edible diploid (2n=22) accessions belonging to the two banana species, Musa acuminata and M. balbisiana. FISH with the 18S-25S probe resulted in signals on one pair of chromosomes, the position of signals corresponded to the secondary constriction at the end of a short arm. The intensity of labelling was different between the homologues and the larger site corresponded to a larger secondary constriction. This labelling pattern was observed consistently in all genotypes. On the other hand, differences in the number of 5S sites were observed between the accessions. While in some of the wild seeded species, the 5S rDNA was localised on two pairs of chromosomes, hybridisation signals appeared on three pairs of chromosomes in other wild accessions. Quite unexpectedly, only five sites of 5S rDNA were reproducibly observed in the two vegetatively propagated diploid edible cultivars, Pisang Mas and Niyarma Yik, evidence for structural heterozygosity. A dual colour FISH showed that in all accessions, the satellite chromosomes carrying the 18S-25S loci did not carry the 5S loci. The results demonstrate that molecular cytogenetics can be applied to Musa and that physical cytogenetic maps can be generated.
The Triticeae species are unique among the important agricultural crops in possessing massive gen... more The Triticeae species are unique among the important agricultural crops in possessing massive genomes with a prevalence of dispersed DNA repeats. The highest level of complexity is observed in tetraploid and hexaploid wheat whose nuclear genomes comprise two and three homoeologous genomes, respectively. Polyploidy and the presence of repeats make gene cloning and genome sequencing in the Triticeae extremely difficult. Chromosome genomics simplifies these tasks by targeting single chromosomes and chromosome arms, which represent only a few percent of the nuclear genomes. The advantages of this strategy over a whole-genome approach include the avoidance of problems due to the presence of homoeologs in wheat, reduction of work to manageable portions, cost efficiency, and an opportunity to structure collaborative projects where individual laboratories work on particular chromosomes. In this chapter, we describe how chromosomes and chromosome arms can be isolated by flow cytometric sorting and we review development of flow cytogenetics in the Triticeae. We then discuss various applications of flow-sorted chromosomes and assess the potential of chromosome genomics in the Triticeae.
BMC Evolutionary Biology, 2011
Background The classification of the Musaceae (banana) family species and their phylogenetic inte... more Background The classification of the Musaceae (banana) family species and their phylogenetic inter-relationships remain controversial, in part due to limited nucleotide information to complement the morphological and physiological characters. In this work the evolutionary relationships within the Musaceae family were studied using 13 species and DNA sequences obtained from a set of 19 unlinked nuclear genes. Results The 19 gene sequences represented a sample of ~16 kb of genome sequence (~73% intronic). The sequence data were also used to obtain estimates for the divergence times of the Musaceae genera and Musa sections. Nucleotide variation within the sample confirmed the close relationship of Australimusa and Callimusa sections and showed that Eumusa and Rhodochlamys sections are not reciprocally monophyletic, which supports the previous claims for the merger between the two latter sections. Divergence time analysis supported the previous dating of the Musaceae crown age to the Cretaceous/Tertiary boundary (~ 69 Mya), and the evolution of Musa to ~50 Mya. The first estimates for the divergence times of the four Musa sections were also obtained. Conclusions The gene sequence-based phylogeny presented here provides a substantial insight into the course of speciation within the Musaceae. An understanding of the main phylogenetic relationships between banana species will help to fine-tune the taxonomy of Musaceae.
Genome, 2003
Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were deve... more Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were developed for rye (Secale cereale L.). Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity obtained after the analysis of DAPI-stained chromosomes (flow karyotypes) were characterized and the chromosome content of the DNA peaks was determined. Chromosome 1R could be discriminated on a flow karyotype of S. cereale 'Imperial'. The remaining rye chromosomes (2R-7R) could be discriminated and sorted from individual wheat-rye addition lines. The analysis of lines with reconstructed karyotypes demonstrated a possibility of sorting translocation chromosomes. Supernumerary B chromosomes could be sorted from an experimental rye population and from S. cereale 'Adams'. Flow-sorted chromosomes were identified by fluorescence in situ hybridization (FISH) with probes for various DNA repeats. Large numbers of chromosomes of a single type sorted onto microscopic slides facilitated detection of rarely occurring chromosome variants by FISH with specific probes. PCR with chromosome-specific primers confirmed the identity of sorted fractions and indicated suitability of sorted chromosomes for physical mapping. The possibility to sort large numbers of chromosomes opens a way for the construction of large-insert chromosome-specific DNA libraries in rye.
Plant Journal, 2004
A novel high-resolution fluorescence in situ hybridisation (FISH) strategy, using super-stretched... more A novel high-resolution fluorescence in situ hybridisation (FISH) strategy, using super-stretched flow-sorted plant chromosomes as targets, is described. The technique that allows longitudinal extension of chromosomes of more than 100 times their original metaphase size is especially attractive for plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves flow cytometric sorting of metaphase chromosomes, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol:acetic acid (3 : 1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea, hybridised with 45S and 5S ribosomal DNAs and the [GAA]n microsatellite, the [TTTAGGG]n telomeric repeat and a bacterial artificial chromosome (BAC) clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity of 1 kbp. Spatial resolution of neighbouring loci was improved down to 70 kbp as compared to 5–10 Mbp after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA fibres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is confined to species for which chromosome flow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.
A novel high-resolution¯uorescence in situ hybridisation (FISH) strategy, using super-stretched¯o... more A novel high-resolution¯uorescence in situ hybridisation (FISH) strategy, using super-stretched¯owsorted plant chromosomes as targets, is described. The technique that allows longitudinal extension of chromosomes of more than 100 times their original metaphase size is especially attractive for plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves¯ow cytometric sorting of metaphase chromosomes, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol:acetic acid (3 : 1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea, hybridised with 45S and 5S ribosomal DNAs and the [GAA] n microsatellite, the [TTTAGGG] n telomeric repeat and a bacterial arti®cial chromosome (BAC) clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity of 1 kbp. Spatial resolution of neighbouring loci was improved down to 70 kbp as compared to 5±10 Mbp after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA ®bres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is con®ned to species for which chromosome¯ow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.